Core domain of annexins and uses thereof in antigen delivery and vaccination

ABSTRACT

The present disclosure provides immunogenic compositions, such as vaccines, including DNA vaccines, and uses thereof, e.g., which include an annexin core domain to mediate efficient antigen delivery and antigen presentation in order to induce an antigen-specific immune response and/or to treat or prevent infectious diseases and/or cancer.

The present disclosure provides immunogenic compositions, such asvaccines, including DNA vaccines, and uses thereof, e.g., which includean annexin core domain for mediating efficient antigen delivery andantigen presentation in order to induce an antigen-specific immuneresponse, and/or to treat or prevent infectious diseases and/or cancer.

BACKGROUND OF THE INVENTION

Central to the initiation of an adaptive immune response areprofessional antigen presenting cells (APC), which displayantigen-derived peptides bound to MHC class I and class II complexes ontheir cell surface (Verboogen, Dingjan et al. 2016). While cytosolicproteins are degraded by the proteasome and loaded onto MHC class Icomplexes recognized by CD8+ T cells, engulfment of exogenous proteins(e.g. from phagocytosed bacteria or apoptotic cells) leads toendosomal/lysosomal degradation and presentation on MHC class IIcomplexes presented to CD4+ T cells. In addition, APC such as dendriticcells (DC) are able to shuttle peptides derived from engulfed proteinsalso into the MHC class I pathway to be presented to CD8+ T cells, aprocess termed cross-presentation (Segura and Amigorena 2015). Amongstdifferent cells types described to fulfill APC-like functions, DC areregarded as the most efficient (Kambayashi and Laufer 2014). FollowingAPC:T cell interactions, T-cell receptors (TCR) engagement leads toinitial T cell activation (priming), characterized, e.g., by secretionof cytokines like Interleukin (IL)-2 and Interferon-γ (Grakoui, Bromleyet al. 1999). Activated T cells will proceed to divide and differentiateinto different types of effector T cells, which can be classified in twomajor lineages, CD4+ T helper cells (Th) and CD8+ cytotoxic T cells.Cytotoxic T cells directly induce apoptosis in target cells, while Thcells direct immune responses by production of cytokines and have beenclassified into Th1, Th2 and Th17 major subsets (Lutz 2016). Summarizingtheir effector functions, Th1 cells are necessary to activate cellularimmunity while Th2 cells induce humoral immune responses. Th17 cells arethought to be involved in immunity against extracellular pathogens likefungi. Regarding anti-tumor immune responses, the induction of efficientCD8+ T cell response has been regarded as critical for tumor rejection,and many tumor vaccination regimes fail to induce CD8+ T cell anti-tumorresponses (Buhrman and Slansky 2013). Thus, efficient antigenpresentation by APCs plays a pivotal role for induction of adaptiveimmunity.

Annexins comprise a family of calcium- and phospholipid-bindingproteins. Over 20 members have been found in all eukaryotic kingdoms aswell as plants and animals with the exception of fungi. Annexins havemolecular weights ranging between 30 and 40 kDa (only annexin VI is 66kDa) and possess striking structural features. Annexins' aminoterminaldomains are diverse in sequence and length (ranging from 11 to 196) oneach annexin member. In contrast the carboxyterminal regions consistingof four (eight only for annexin VI) a-helical domains composed of about70 amino acid residues are well conserved among annexins. The calcium-and phospholipid-binding sites are located in the carboxyterminaldomains. The Ca²⁺ binding similarities of all the annexins is due totheir common primary structure, a unique N-terminal domain (the “tail”)and the conserved C-terminal domain (the “core”). With the exception ofannexin VI, the conserved C-terminal domain is always composed of 4repeats (annexin VI having 8) of −70 amino acids containing an increasedhomology region called the “endonexin fold”. In addition to the Cterminal core the annexins contain a significantly more variable Nterminal head. It is this domain which endows each annexin with uniquefunctions in a diverse range of cellular processes including; endo- andexocytosis, cytoskeletal regulation and membrane conductance andorganisation. Given their involvement in such a variety of processes itis not surprising that the annexins have also been implicated in a rangeof disease pathologies. Although there is no singular disease statedirectly attributed to a dysregulation in annexin function, severalpathological conditions are suggested to be modified by the annexins.Fatimathas and Moss (Fatimathas and Moss 2010) discuss the growingevidence for the role of the annexins in the progression of cancer,diabetes and the autoimmune disorder anti-phospholipid syndrome.

In all annexins, lipid binding is mediated by the C-terminal core domainhighly conserved among all annexin family members (Gerke and Moss 2002,Moss and Morgan 2004). In contrast, annexin N-termini vary in sequence.Peptides corresponding to the AnxA1 N-terminus were shown to bind tomembers of the N-formyl peptide receptor (FPR) family, resulting in areduction of neutrophil transmigration in several models of acute andchronic inflammation (Walther, Riehemann et al. 2000, Strausbaugh andRosen 2001, Ernst, Lange et al. 2004, Perretti and Dalli 2009).Downstream signaling induced by binding of AnxA1 N-terminal peptides toFPR family members causes activation of ERK, but not of p38 or JNK(Hayhoe, Kamal et al. 2006, Pupjalis, Goetsch et al. 2011). The presenceof multiple annexin family members in all higher eukaryotes suggests afundamental role for annexins in cell biology. Mice deficient inindividual annexin family members, however, have no severe phenotype,suggesting that several annexins have (partly) overlapping functions(Gerke and Moss 2002, Farber, De Rose et al. 2003). In fact, functionalredundancy of annexins was proven in the context of membranetrafficking, inhibition of PLA₂ activity and blood coagulation (Gerkeand Moss 2002).

US 2002-052358 describes a method of treating a subject with arthritisor an arthritic disease or preventing arthritis or arthritic disease ina subject, comprising administering to the subject a therapeuticallyeffective amount of an agent that attenuates annexin function. Alsoprovided are various methods of screening for agents.

WO 01/10199 describes a knockout transgenic mouse containing anonfunctional allele of the tumor suppressing gene, annexin VII. Thismouse is used as a screening model for potential therapeutic agentsuseful in the treatment of tumors resulting from an annexin tumorsuppressor disease.

JP 2014-095643 describes screening of a compound effective in treatmentof inflammatory disease, based on an inhibition of binding betweenannexin A2 and ADAM17.

WO 2014/126127 describes a method for screening an active ingredient forthe treatment of severe enanthema, skin erythema, body surface erosion,blister and excoriation as formyl peptide receptor 1-inducednecroptosis-related diseases. The active ingredient to be screened issaid to be a substance capable of inhibiting necroptosis that is inducedby the binding of formyl peptide receptor 1 to annexin A1.

WO 02/17857 discloses methods for inhibiting angiogenesis in endothelialcells and selectively inducing apoptosis in endothelial cells viacompounds which binds annexin II are provided. These compounds andmethods for using these compounds are regarded as useful in thetreatment of diseases or disorders characterized by unwantedangiogenesis. Also provided are pharmaceutical compositions containing acompound which binds annexin II and a pharmaceutically acceptablevehicle and methods for identifying such compounds.

WO 2005/027965 discloses anti-annexin antibodies and their uses as wellas uses of theirs ligands, the annexins. Such annexins and anti-annexinantibodies are useful for detecting apoptosis and for the production ofpharmaceutical compositions for the diagnosis and/or treatment ofcancer, autoimmune diseases, cardiovascular and/or vascular diseases.

US 2014/0322214 discloses includes compositions and methods for bindingDectin-1 on immune cells with anti-Dectin-1-specific antibodies orfragment thereof capable of activating the immune cells as well asmethods for treating or preventing an influenza infection in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising ananti-dectin-1 antibody fused to an influenza antigen. The thesis ofConnie Hesse, CLEC7A/Dectin-1 attenuates the immune response againstdying and dead cells, Friedrich-Alexander-University Erlangen-Nürnberg,2011, discusses the role of C-type lectins CLEC4L/DC-SIGN, CLEC9A/DNGR1,and CLEC7A/dectin-1 in the recognition as well as the uptake ofapoptotic and necrotic cells and/or their effects on the immunogenicityof dying and dead cells.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is amembrane receptor displaying both scavenging and signaling functions.The wide variety of extracellular ligands and of cytoplasmic scaffoldingand signaling proteins interacting with LRP-1 gives it a major role notonly in physiological processes, such as embryogenesis and development,but also in critical pathological situations, including cancer andneurological disorders (Emonard, Theret et al. 2014). Cell surfaceannexin VI may function as an acidic pH binding site or receptor and mayalso function as a co-receptor with LRP-1 at neutral pH in the contextof alpha 2-macroglobulin recognition (Ling, Chen et al. 2004).

Arur and colleagues (Arur, Uche et al. 2003) as well as Tzelepis et al.(Tzelepis, Verway et al. 2015) describe a role for annexin A1 in theprocess of phagocytosis of apoptotic cells, which is regarded asimmunologically silent and not leading to a T cell response. In the samepublication, Tzelepis and colleagues further described a role forendogenous annexin A1 in the process of cross presentation. Thispublication describes annexin A1 as a mediator that acts in the cytosolof dendritic cells. Therefore, this publication does not enable the useof the annexin core domain as exogenous mediator to engage antigenpresentation and cross presentation.

Andersen and colleagues (Andersen, Xia et al. 2016) describe the bindingof annexin A2 to Toll-like receptor (TLR) 2. By triggering TLR2, annexinA2 can act as a vaccine adjuvant, enhancing TLR-mediated DC activationand processes like upregulation of co-stimulatory surface molecules andantigen cross-presentation. This publication is silent about antigendelivery into DC.

Tzelepis et al. (in: Tzelepis et al. Annexin1 regulates DC efferocytosisand cross-presentation during Mycobacterium tuberculosis infection. JClin Invest. 2015 February; 125(2):752-68. Epub 2014 Dec. 22) disclosethat during Mycobacterium tuberculosis (Mtb) infection, the engulfmentligand annexin1 is an important mediator in DC cross-presentation thatincreases efferocytosis in DCs and intrinsically enhances the capacityof the DC antigen-presenting machinery. Annexin1-deficient mice werehighly susceptible to Mtb infection and showed an impaired Mtbantigen-specific CD8+ T cell response.

Finally, Weyd and colleagues (Weyd, Abeler-Dorner et al. 2013, Linke,Abeler-Dorner et al. 2015) disclose that in mice, Annexin A1, AnnexinA5, Annexin A13 and the annexin core domain prevented the development ofinflammatory DC and suppressed the cellular immune response against themodel antigen ovalbumin (OVA) expressed in apoptotic cells.

Reagents which react specifically or preferentially with DC and mediateantigen presentation have great potential as targeting agents to inducepotent immune responses to tumor or infectious disease antigens. Thesecell-specific targeting agents could also be engineered to delivertoxins to eliminate potent antigen presenting cells (e.g., DC) in bonemarrow and organ transplantations or other autoimmune disorders.Accordingly, such DC-specific binding agents possess great therapeuticand diagnostic value.

It is therefore an object of the present invention to provide such newreagents and to employ these reagents in the development of new andeffective therapies. Other objects and aspects of the present inventionwill become apparent to the person of skill upon reading the followingdescription of the invention.

The invention pertains to an isolated annexin core domain, the annexincore domain being defined to comprise an amino acid sequence of anannexin core domain shown within the sequence selected from the group ofSEQ ID Nos. 1 to 3 and 6 to 8, or to comprise an amino acid sequencethat is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, most preferably99% identical to an annexin core domain amino acid sequence as comprisedwithin a sequence selected from the group of SEQ ID Nos. 1 to 3 and 6 to8. The definitions of the core domains is provided herein below in theexample section. In particular preferred is an annexin core domainconsisting of an amino acid sequence of a core domain as shown in anamino acid sequence selected from the group of SEQ ID Nos. 1 to 3 and 6to 8; or consisting of an amino acid sequence that is at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 98%, most preferably 99% identical to an aminoacid sequence of a core domain as shown in an amino acid sequenceselected from the group of SEQ ID Nos. 1 to 3 and 6 to 8.

The present invention also provides a protein conjugate or fusionprotein comprising (i) at least one annexin core domain as describedherein, and (ii) at least one antigenic peptide that can be presented byMHC (preferably HLA). The antigenic peptide can be derived from a tumoror infectious agent, pathogen or endogenous protein. The presentinvention also provides respective vaccines comprising fusions and/orconjugates and other therapeutic compositions.

In certain embodiments the protein conjugate or fusion protein of theinvention comprises a covalent linkage between the annexin core domainand the at least one antigenic peptide that can be presented by MHC.Also encompassed are conjugates or fusion protein of the invention wherethe covalent linkage includes a linker molecule or peptide. Selection ofsuitable linker molecules is well established in the pertinent art. Insome embodiments of the invention the linker comprises an amino acidsequence of the linker as shown in FIG. 10 (SEQ ID NO: 15), or asequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to the linker sequence shown in FIG. 10B (SEQ ID NO:15).

In some aspects and embodiments the present invention the fusion proteinis encoded by the nucleic acid shown in SEQ ID NO: 13, or a by a nucleicacid variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 13. Preferably the fusion proteinof the invention comprises the amino acid sequence of SEQ ID NO: 14, orof a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 14.

The term “fusion protein” as used herein relates to an artificialproteinaceous construct and means a protein comprising at least twodifferent amino acid sequences which are defined by their origin and/orby special functions. In this aspect the fusion protein of the inventioncomprises the annexin core domain amino acid sequence fused to secondamino acid sequence of another protein which is not annexin, and whichis antigenic in the sense that said second protein or fragments thereof,are presented on a cell via the MHC complex. Moreover, the term fusionprotein according to the present invention does further include suchfusion proteins which also contain non-protein molecules such as nucleicacids, sugars, or markers for radioactive or fluorescent labelling.

It was surprisingly found that the constructs according to the presentinvention have an immune stimulating (enhancing) effect. Thus, thecompositions of the invention containing the annexin core domain complexand/or fusion can be used in a variety of DC-targeted therapies, forexample, to enhance antigen presentation and/or induce T cell responses,such as cytotoxic T cell (CTL) responses, against a variety of targetcells or pathogens, or to treat antigen presenting cell (APC)-mediateddiseases. The invention surprisingly found that combining an antigenicmolecule, such as an antigenic peptide, with an annexin core domain asdescribed herein, significantly enhances the immune modulatory effectsof said antigenic sequence. Without being bound to a particular theory,coupling an annexin core domain to an antigenic molecule enhancesantigen processing and MHC-presentation of antigen presenting cells suchas dendritic cells. Therefore the invention broadly enables a productsand methods for enhancing the antigen presentation of antigenicmolecules via MHC, preferably human MHC (HLA).

As used herein, the term “antigen” refers to a substance capable ofeliciting an immune response, e.g., a T-cell-mediated immune response bythe presentation of the antigen on Major Histocompatibility Antigen(MHC) cellular proteins and causing an antigen-specific T-cellsresponse. In the case of a regulatory T-cell (Treg) response to theantigen is a decrease or amelioration of the immune response by othereffector cells, e.g., helper T-cells (Th) and/or cytotoxic T-cells (Tc).The skilled immunologist will recognize that when discussing antigensthat are processed for presentation to T-cells, the term “antigen”refers to those portions of the antigen (e.g., a peptide fragment) thatis a T-cell epitope presented by MHC to the T-cell receptor. When theexpression “antigen” is modified by self- or auto-, this refers to selfor auto antigens that are commonly present in MHC molecules but thatalso trigger a T-cell response. When used in the context of a B cellmediated immune response in the form of an antibody that is specific foran “antigen”, the portion of the antigen that binds to thecomplementarity determining regions of the variable domains of theantibody (light and heavy) the bound portion may be a linear orthree-dimensional epitope. In certain cases, the antigens delivered bythe vaccine or fusion protein or protein conjugate of the presentinvention are internalized and processed by antigen presenting cellsprior to presentation, e.g., by cleavage of one or more portions of theantibody or fusion protein.

As used herein, the term “antigenic peptide” refers to that portion of apolypeptide antigen that is specifically recognized by either B-cellsand/or T-cells. B-cells respond to foreign antigenic determinants viaantibody production, whereas T-lymphocytes mediate cellular immunity.Thus, antigenic peptides in a T-cell response are those parts of anantigen that are recognized by antigen-specific T-cell receptors in thecontext of MHC.

As used herein, the term “epitope” refers to any protein determinantcapable of specific binding to an immunoglobulin or of being presentedby a Major Histocompatibility Complex (MHC) protein (e.g., Class I orClass II) to a T-cell receptor. Epitopic determinants are generallyshort peptides 5-30 amino acids long that fit within the groove of theMHC molecule that presents certain amino acid side groups toward theT-cell receptor and has certain other residues in the groove, e.g., dueto specific charge characteristics of the groove, the peptide sidegroups and the T-cell receptor. Generally, an antibody specificallybinds to an antigen when the dissociation constant is 1 mM, 100 nM oreven 10 nM.

As used herein the term “Antigen Presenting Cells” (APC) are cells thatare capable of activating T-cells, and include, but are not limited to,certain macrophages, B cells and dendritic cells. “Dendritic cells”(DCs) refer to any member of a diverse population of morphologicallysimilar cell types found in lymphoid or non-lymphoid tissues. Thesecells are characterized by their distinctive morphology, high levels ofsurface MHC-class II expression (Steinman, et al., Ann. Rev. Immunol.9:271 (1991); incorporated herein by reference for its description ofsuch cells). These cells can be isolated from a number of tissuesources, and conveniently, from peripheral blood or differentiated frommurine bone marrow, as described herein. Dendritic cell binding proteinsrefer to any protein for which receptors are expressed on a dendriticcell. Examples include GM-CSF, IL-1, TNF, IL-4, CD40L, CTLA4, CD28, andFLT-3 ligand. An antigenic peptide comprises a peptide sequence that iscapable to be presented by HLA molecules (MHC class I and/or MHC classII) and induces a T cell response, such as cytotoxic T cell (CTL)response. Usually, these peptides are between 8 and 30, preferablybetween 8 and 24 amino acids long, MHC class I peptides are usuallybetween 8 and 10 long, and MHC class II peptides are usually between 21and 25 amino acids long. Methods to identify (“screen”) for theseantigenic peptides are known as well and can involve both in vivo or invitro and in silico methods.

Methods to prepare respective conjugates (i.e. comprising non-covalentor covalent bonds introduced between different components, i.e. theannexin and the peptide) of the annexin core domain and the antigenicpeptide as well as to prepare respective fusion proteins (i.e.expression of one protein after recombinant cloning of the components)are well known in the art.

In the context of the present invention, the term “annexin core domain”shall be understood as indicating/representing the minimal fragment ofthe polypeptide for annexin (or homologs thereof), which is necessaryand sufficient to mediate antigen presentation (see also below). Somepreferred proteinaceous annexin core domains are defined herein above.This ability (biological function) may be tested in a number of artknown methods as described herein, and, e.g. in the examples, below.This ability may further be tested in a number of art known methods asdescribed in the respective literature. For examples of annexin coredomains, see also FIG. 7, below. Also, the term shall particularlycomprise the vertebrate, in particular mammalian (in particular human)annexin gene and/or protein and/or mRNA and/or the core fragment (coredomain) as described herein. The term also covers the annexin coredomain in different preparations, such as in the cellular context, acell recombinantly expressing said core domain, purified from the cell,and fractions, in particular biologically active factions, thereof.

Protein aggregates are known to enhance immune responses. The mechanismby which protein aggregates mediate such potent antibody responses isnot fully understood. However, it is believed that the potency is due,at least in part, to the ability of the multivalent protein toextensively cross link the cell surface receptors such asimmunoglobulins of B cells and activate the B cells. Therefore it is incontext of the invention one embodiment to aggregate the proteinconjugate or fusion protein of the invention to further enhance immuneresponses. This may be achieved by using multimeric antigenic peptideswhere the antigenic molecule is multimerized directly or via a linkersequence to form a poly-antigenic peptide with a repeating antigenicsequence for fusion with the annexin core domain in accordance with theinvention. Alternatively the fusion protein of the invention may furthercomprise a moiety that induces aggregation of the protein conjugate orfusion protein, such as a protein multimerization domain or dimerizationdomain, which is covalently attached to the fusion protein. Oneparticularly favorable example of such a protein multimerization domainis a coiled-coil domain, such as an isoleucine zipper domain thatpromotes trimerization of multiple polypeptides having such a domain. Afurther favorable example of a modification for protein multimerizationis the use of conjugated biotin or a biotinylation sequence inconjunction with the protein streptavidin. Another option in context ofthe invention provides compositions of the fusion protein of theinvention in combination with the agent for protein aggregation.

Fusion proteins can also be made at the nucleic acid coding level byplacing, in-line and in the correct coding frame, the two or moresequences of the portions of the proteins or peptides, i.e. of theannexin core domain and the respective antigenic peptide or antigen.Fusion proteins are synthesized by methods known to those of skill inthe art including, e.g., solid phase protein synthesis, and by moleculartechniques that permit the manipulation of DNA in vitro, includingpolymerase chain reaction (PCR) and oligonucleotide-directedmutagenesis.

In the context of the present invention, the terms “C-type lectinreceptor”, “Dectin-1”, “DC-SIGN”, and “LRP-1” shall be understood asindicating/representing the minimal fragment of the receptor(s), whichis necessary and sufficient to bind to a core domain of the annexin asdescribed and tested in the examples, and in, for example, Hesse asmentioned above for lectin-Fc fusion proteins. This ability may furtherbe tested in a number of art known methods as described in therespective literature. Also, the term shall comprise the mammalian (inparticular mouse) homo log of the human receptor gene and/or proteinand/or mRNA and/or the fragment (binding part, fragment or domain) asdescribed herein. The term also covers the receptor(s) and/or theminimal fragment of the receptor(s) in different preparations, such asin the cellular context, a cell (recombinantly) expressing saidreceptor(s) and/or the minimal fragment of the receptor(s), purifiedfrom the cell, and fractions thereof.

With Dectin-1 and DC-SIGN as members of the family of C-type lectinreceptors and LRP-1, novel DC-surface receptors could be identified thatwith high affinity bind to the core domain of all annexins as studied.This is an indication that Dectin-1, DC-SIGN and LRP-1 are responsiblefor the annexin-mediated effects on the immune response and induction ofantigen presentation.

The effect of the annexins on DC via specific receptors is a novelmolecular mechanism of antigen presentation, resulting in a multitude ofnovel possibilities both for the therapy of cancers and tumors, as wellas for infectious diseases in mammals, such as mice and humans.

WO 2009/049892 describes a first polypeptide (A) comprising a recruitingpolypeptide (a) comprising at least an annexin core domain or afunctional variant thereof, a bait polypeptide (b) and a luminophore.The composition according to the invention can be used to measureprotein-protein interactions within and/or between entire multiproteincomplexes. Described is the use of the method according to the inventionfor the identification of a test compound in a library of test compoundswhich modulates a medically relevant protein-protein interaction,without that any concrete disease context is disclosed. WO 2009/049892is silent about any interaction(s) of annexin with Dectin-1, DC-SIGNand/or LRP-1, and also non-enabling for the screening of therapeuticallyrelevant compounds and/or compositions.

WO 2005/027965 describes that annexin I and other annexins are relatedto specific receptors, which could be stimulated or blocked by eitherbinding of one of the annexins or fragments thereof or an antibodyagainst this receptor. Thus, annexins and/or functional fragmentsthereof and/or fusion proteins comprising an annexin or functionalfragments thereof are discussed to be of use to modulate the immunesystem. WO 2005/027965 is silent about the use of the annexin coredomain itself to mediate antigen presentation, and thus is alsonon-enabling for the screening of therapeutically relevant compoundsand/or compositions.

Exposure of bone marrow-derived DC (BMDC) to a fusion protein comprisingthe annexin core domain and the model antigen ovalbumin (OVA) in vitroresulted in profound antigen presentation of OVA-derived peptides insurface MHC class I molecules (FIG. 1) as well as in strongly amplifiedspecific T cell stimulation of both, CD8+ and CD4+ T cells (FIGS. 2 and3). These results suggest that the annexin core domain has a previouslyunappreciated role in antigen presentation and antigencross-presentation. Manipulating anx-core-domain mediated antigenpresentation may, therefore, prove useful when designing vaccinationstrategies and, accordingly, beneficial for patients with cancer(vaccination with tumor antigens) or infectious diseases. Of note, thismechanism, in which the annexin core domain mediates antigen deliveryand antigen presentation when administered exogenously to DC and linkedto an antigen is inherently different from described endogenous,cytosolic functions of annexin A1 (Tzelepis, Verway et al. 2015). Thismechanism is also different from described vaccine adjuvant function ofannexin A2 binding to Toll-like receptor 2 (Andersen, Xia et al. 2016),because the annexin core domain as described here does not mediate DCstimulation via TLRs but mediates antigen delivery and antigen (cross-)presentation.

Preferred is a protein conjugate or fusion protein according to thepresent invention, wherein said antigenic peptide is derived from aprotein selected from the group consisting of βhCG, gp100 or Pmel17,HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, NY-BR-1, NY-CO-58,MN (gp250), idiotype, Tyrosinase, Telomerase, SSX2, MUC-1, MART1,melan-A, NY-ESO-1, MAGE-1, MAGE-3, MAGE-A3, and high molecularweight-melanoma associated antigen (HMW-MAA).

Other antigenic peptides for use with the present invention includecancer peptides selected from tumor-associated antigens, e.g.,autologous cancer antigens obtained from a patient. Non-limitingexamples of cancer antigens include antigens from leukemias andlymphomas; neurological tumors such as astrocytomas or glioblastomas;melanoma; breast cancer; lung cancer; head and neck cancer;gastrointestinal tumors; gastric cancer; colon cancer; liver cancer;pancreatic cancer; genitourinary tumors such cervix; uterus; ovariancancer; vaginal cancer; testicular cancer; prostate cancer or penilecancer; bone tumors; vascular tumors; or cancers of the lip;nasopharynx; pharynx and oral cavity; esophagus; rectum; gall bladder;biliary tree; larynx; lung and bronchus; bladder; kidney; brain andother parts of the nervous system; thyroid; Hodgkin's disease;non-Hodgkin's lymphoma; multiple myeloma and leukemia. In a specificaspect the composition further comprises antigenic peptides selectedfrom tumor associated antigens are selected from CEA; prostate specificantigen (PSA); HER-2/neu; BAGE; GAGE; MAGE 1-4; 6 and 12; MUC (Mucin)(e.g.; MUC-1, MUC-2, etc.); GM2 and GD2 gangliosides; ras; myc;tyrosinase; MART (melanoma antigen); MARCO-MART; cyclin B1; cyclin D;Pmel 17(gp100); GnT-V intron V sequence (N-acetylglucoaminyltransferaseV intron V sequence); Prostate Ca psm; prostate serum antigen (PSA);PRAME (melanoma antigen); β-catenin; MUM-1-B (melanoma ubiquitousmutated gene product); GAGE (melanoma antigen) 1; BAGE (melanomaantigen) 2-10; C-ERB2 (Her2/neu); EBNA (Epstein-Barr Virus nuclearantigen) 1-6; gp75; human papilloma virus (HPV) E6 and E7; p53; lungresistance protein (LRP); Bcl-2; and Ki-67.

Further antigenic peptides or antigens for use in context with thepresent invention are selected from viral antigens. The term “viralantigen” includes any substance that elicits an immune response againsta virus. Examples include Retro viridae, in particular HIV-I and HIV-LP;Picornaviridae, in particular polio virus and hepatitis A virus;enterovirus, in particular human coxsackie virus, rhinovirus, echovirus;Calciviridae, in particular strains that cause gastroenteritis;Togaviridae, in particular equine encephalitis virus and rubella virus;Flaviridae, in particular dengue virus, encephalitis virus and yellowfever virus; Coronaviridae, in particular coronavirus; Rhabdoviridae, inparticular vesicular stomatitis virus and rabies virus; Filoviridae, inparticular Ebola virus or and Marburg virus; Paramyxoviridae, inparticular parainfluenza virus, mumps virus, measles virus andrespiratory syncytical virus; Orthomyxoviridae, in particular influenzavirus; Bungaviridae, in particular Hantaan virus, bunga virus,phlebovirus and Nairo virus; Arena viridae, in particular hemorrhagicfever virus; Reoviridae, in particular reovirus, orbivirus androtavirus; Birnaviridae; Hepadnaviridae, in particular Hepatitis Bvirus; Parvovirida, in particular parvovirus; Papovaviridae, inparticular papilloma virus, simian virus-40 (SV40) and polyoma virus;Adenoviridae; Herpesviridae, in particular herpes simplex virus (HSV) 1and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus;Poxyiridae, in particular variola virus, vaccinia virus and pox virus;and Irido viridae, in particular African swine fever virus; Hepatitis C,and HPV L6, HPV L7, fragments and derivatives thereof.

Further antigenic peptides or antigens for use in context with thepresent invention are selected from bacterial antigens. As used herein,the term “bacterial antigen” includes any substance that elicits animmune response against a bacterium. Examples include Helicobacterspecies, in particular Helicobacter pyloris; Borelia species, inparticular Borelia burgdorferi; Legionella species, in particularLegionella pneumophilia; Mycobacteria species, in particular M.tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae;Staphylococcus species, in particular Staphylococcus aureus; Neisseriaspecies, in particular N. gonorrhoeae, N. meningitidis; Listeriaspecies, in particular Listeria monocytogenes; Streptococcus species, inparticular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S.pneumoniae; anaerobic Streptococcus species; pathogenic Campylobacterspecies; Enterococcus species; Haemophilus species, in particularHaemophilus influenzae; Bacillus species, in particular Bacillusanthracis; Corynebacterium species, in particular Corynebacteriumdiphtheriae; Erysipelothrix species, in particular Erysipelothrixrhusiopathiae; Clostridium species, in particular C. perfringens, C.tetani; Enterobacter species, in particular Enterobacter aerogenes,Klebsiella species, in particular Klebsiella pneumoniae, Pasteurellaspecies, in particular Pasteurella multocida, Bacteroides species;Fusobacterium species, in particular Fusobacterium nucleatum;Streptobacillus species, in particular Streptobacillus moniliformis;Treponema species, in particular Treponema pertenue; Leptospira;pathogenic Escherichia species; and Actinomyces species, in particularActinomyces israelii.

Preferred is furthermore a protein conjugate or fusion protein accordingto the present invention, wherein said conjugate or said fusion proteinis further conjugated/fused to a co-stimulatory molecule or animmunogenic fragment thereof or a costimulatory second peptide sequence.

Another aspect of the present invention then relates to a nucleic acidencoding for the fusion protein or protein conjugate according to thepresent invention. Preferably, the coding sequence codes for an antigenderived from a protein selected from the group consisting of βhCG, gp100or Pmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, NY-BR-1,NY-CO-58, MN (gp250), idiotype, Tyrosinase, Telomerase, SSX2, MUC-1,MART1, melan-A, NY-ESO-1, MAGE-1, MAGE-3, MAGE-A3, and high molecularweight-melanoma associated antigen (HMW-MAA). More preferably, saidcoding sequence is fused to at least one (additional) DC-stimulatorynucleic acid sequence. It is also possible to use fusions of multipleantigenic peptides, for example multiple sequences found in one tumordisease, or patient specific antigens found in an individual tumor.

Another aspect of the present invention then relates to a recombinantexpression vector expressing the nucleic acid according to theinvention.

The invention also relates to an isolated annexin core domain comprisingan amino acid sequence of the core domain as shown in the sequencesselected from SEQ ID Nos. 1 to 3 and 6 to 8. The domain ranges areprovided herein in the example section. Preferred are domains consistingof said sequences, or essentially consist of said sequences (e.g. having5 to 10 amino acid extensions that do not interfere with the function ofthe domain). The annexin core domain according to the present inventionthat can further be used in the method according to the presentinvention can be derived from any of the known annexins or functionalfragments (i.e. able to bind to the receptors as described herein)thereof, and is preferably selected from the group of the human ormurine annexin 1, 5, and 13 core domain, preferably according to asequence comprised in the sequence according to SEQ ID NO: 1 to 3 and 6to 8, or according to a sequence of a core comprised in the sequenceaccording to SEQ ID NO: 1, 2, 3, 6, 7, or 8, or functional fragmentsthereof, more preferably according to the boxed sequences as shown inFIG. 8.

The term “contact” in the present invention means any interactionbetween the potentially binding substance(s)/antigens with the annexincore domain, whereby any of the two components can be independently ofeach other in a liquid phase, for example in solution, or in suspensionor can be bound to a solid phase, for example, in the form of anessentially planar surface or in the form of particles, pearls or thelike.

Another aspect of the present invention relates to a method formanufacturing a pharmaceutical composition for treating or preventinginfectious diseases or cancer, comprising the step of admixing theprotein conjugate or fusion protein according to the present invention,or the nucleic acid according to the present invention, or theexpression vector according to the present invention, with a suitableagent or carrier.

Thus, the compounds of the invention can be admixed with suitableauxiliary substances and/or additives. Such substances comprisepharmacological acceptable substances, which increase the stability,solubility, biocompatibility, or biological half-life of the interactingcompound or comprise substances or materials, which have to be includedfor certain routs of application like, for example, intravenoussolution, sprays, band-aids or pills.

Carriers, excipients and strategies to formulate a pharmaceuticalcomposition, for example to be administered systemically or topically,by any conventional route, in particular enterally, e.g. orally, e.g. inthe form of tablets or capsules, parenterally, e.g. in the form ofinjectable solutions or suspensions, topically, e.g. in the form oflotions, gels, ointments or creams, or in nasal or a suppository formare well known to the person of skill and described in the respectiveliterature.

Another aspect of the present invention thus is a pharmaceuticalcomposition comprising the protein conjugate or fusion protein accordingto the present invention, or the nucleic acid according to the presentinvention, or the expression vector according to the present invention.Preferably, the pharmaceutical composition is a vaccine.

Administration of an agent, e.g., the complex or fusion, can beaccomplished by any method which allows the agent to reach the targetcells. These methods include, e.g., injection, deposition, implantation,suppositories, oral ingestion, inhalation, topical administration, orany other method of administration where access to the target cells bythe agent is obtained. Injections can be, e.g., intravenous,intradermal, subcutaneous, intramuscular or intraperitoneal.Implantation includes inserting implantable drug delivery systems, e.g.,microspheres, coated microparticles, hydrogels, polymeric reservoirs,cholesterol matrices, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused orpartially fused pellets. Suppositories include glycerin suppositories.Oral ingestion doses can be enterically coated. Inhalation includesadministering the agent with an aerosol in an inhalator, either alone orattached to a carrier that can be absorbed. The agent can be suspendedin liquid, e.g., in dissolved or colloidal form. The liquid can be asolvent, partial solvent or non-solvent. In many cases, water or anorganic liquid can be used.

In certain embodiments, the compound (activator or inhibitor) isadministered to the subject by administering a recombinant nucleic acid,such as, for example, an annexin core domain or antigen RNA. Preferably,the recombinant nucleic acid is a gene therapy vector.

Another aspect of the present invention relates to a method or use asdescribed herein, wherein the pharmaceutical composition furthercomprises additional pharmaceutically active ingredients for treating orpreventing autoimmune diseases, chronic inflammatory diseases, allergiesor cancer, i.e. chemotherapeutics.

Another aspect of the present invention relates to an isolated annexincore domain; a complex or fusion of an annexin core domain with at leastone antigen; an activating antibody, optionally coupled to at least oneantigen or allergenic compound; or a pharmaceutical compositionaccording to the present invention for use in the prevention and/ortherapy of diseases as described herein (see, e.g., below). Preferred isthe complex or fusion for use according to the present invention,wherein said complex or fusion is soluble or bound to a carrier, such asa liposome or latex bead.

Another aspect of the present invention then relates to a method fortreating or preventing infectious diseases or cancer in a patient,comprising administering to said patient an effective amount of anisolated annexin core domain; a complex or fusion of an annexin coredomain with at least one antigen or allergenic compound; an activatingantibody, optionally coupled to at least one antigen or allergeniccompound; or a pharmaceutical composition obtained by the methodaccording to the present invention.

In general, the attending physician will base a treatment on thecompound as identified, and optionally also on other individual patientdata (clinical data, family history, DNA, etc.), and a treatment canalso be performed based on the combination of these factors. This methodof the present invention for example involves integrating individualdiagnostic immunological data with patient clinical information andgeneral healthcare statistics to enable, for example, the application ofpersonalized medicine to the patient. Significant information about drugeffectiveness, drug interactions, and other patient status conditionscan be used, too.

Preferred is a therapeutic method according to the present invention,wherein said mammal to be treated is a mouse, rat or human.

Preferably, an active agent of the invention (preferably the annexincore domain or the protein conjugate or fusion protein of the invention)is administered in form of a pharmaceutical composition comprising anactivating agent as described above, such as an antibody, nucleotide oran activating binding compound for the annexin core domain/receptorbinding. Preferably, said patient is a human being. Treating is meant toinclude, e.g., preventing, treating, reducing the symptoms of, or curingthe disease or condition, i.e. immunological diseases such asimmunodeficiency, infectious diseases or cancer.

In general, the attending physician will base a treatment on thecompound as identified, and optionally also on other individual patientdata (clinical data, family history, DNA, etc.), and a treatment canalso be performed based on the combination of these factors. This methodof the present invention for example involves integrating individualdiagnostic cancer data with patient clinical information and generalhealthcare statistics to enable, for example, the application ofpersonalized medicine to the patient. Significant information about drugeffectiveness, drug interactions, and other patient status conditionscan be used, too.

Preferred is a therapeutic method according to the present invention,wherein said mammal to be treated is a mouse, rat or human.

More preferably, the cancer to be treated is a solid tumor, such as, forexample, selected from breast, bone, ovarian, liver, kidney, and lungcancer.

Preferably, an active agent is administered in form of a pharmaceuticalcomposition, such as a protein conjugate or fusion protein of theinvention, said patient is a human being. Treating is meant to include,e.g., preventing, treating, reducing the symptoms of, or curing thedisease or condition, i.e. cancer. Treatment generally involves theadministration of a therapeutically effective amount of the proteinconjugate or fusion protein of the invention to the subject in need ofthe treatment.

In another aspect the invention provides a method for the vaccination ofa subject comprising the administration of the protein conjugate orfusion protein of the invention to the subject in need of vaccination.The protein conjugate or fusion protein of the invention is preferablyin the form of a vaccine composition and comprises additionally at leastone carrier and/or excipient and/or vaccine adjuvant.

The herein disclosed pharmaceutical and in particular vaccinecompositions preferably further comprise one or more immune stimulatorycompounds such as adjuvants. An “adjuvant” is an agent that enhances theproduction of an immune response in a non-specific manner. Commonadjuvants include suspensions of minerals (alum, aluminum hydroxide,aluminum phosphate) onto which the fusion protein of the invention isadsorbed; emulsions, including water-in-oil, and oil-in-water (andvariants thereof, including double emulsions and reversible emulsions),liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids(such as CpG oligonucleotides), liposomes, Toll-like Receptor agonists(particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and variouscombinations of such components.

An “effective amount” is an amount of the compound(s) or thepharmaceutical composition as described herein that increases antigenpresentation. The amount alleviates symptoms as found for the diseaseand/or condition.

The invention also includes a method for treating a subject at risk forinfectious diseases or cancer, wherein a therapeutically effectiveamount of an annexin core domain conjugate is provided. Being at riskfor the disease can result from, e.g., a family history of the disease,a genotype which predisposes to the disease, or phenotypic symptomswhich predispose to the disease.

The mammalian patient can be a rat, mouse, goat, rabbit, sheep, horse,monkey or human, preferred is a mouse, rat or human.

Yet another preferred aspect of the present invention then relates to akit, comprising materials for vaccination according to the presentinvention as described herein, in one or separate containers, preferablycomprising a screening tool according to the present invention.Optionally, the kit comprises instructions for performing a methodaccording to the present invention as described herein.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The container may be formed from a variety ofmaterials such as glass or plastic. Preferably the kit and/or containercontain/s instructions on or associated with the container thatindicates directions for reconstitution and/or use.

Preferred is a kit according to the present invention, wherein said kitcomprises materials for a method selected from the group of Westernblots and/or Enzyme-Linked Immunosorbent Assay (ELISA). For example, thelabel may indicate that the lyophilized formulation is to bereconstituted to certain antibody concentrations as suitable for theabove methods, such as ELISA.

Further preferred is the use according to the present invention, whereinsaid kit comprises monoclonal antibodies or fragments thereof specificfor the annexin core domain and/or functional parts and variants thereofas described herein.

The following figures, sequences, and examples merely serve toillustrate the invention and should not be construed to restrict thescope of the invention to the particular embodiments of the inventiondescribed in the examples. All references as cited herein are herebyincorporated in their entirety by reference.

FIG. 1 shows that a fusion protein containing the annexin core domainand the model antigen ovalbumin (Anx-OVA) leads to strongly enhancedantigen cross-presentation in MEW class I molecules on dendritic cells(DC) compared to the antigen OVA alone. A) Schematic presentation of theexperiment. Murine bone marrow derived DC were incubated with OVA orAnx-OVA. Cross presented OVA-derived peptide SIINFEKL (SEQ ID NO: 4)within MEW I molecules on DC was detected by a specific antibody(anti-MHC-SIINFEKL (SEQ ID NO: 4), antibody 25-D1.16, eBioscience). B)Representation of DC positive for cross presented OVAderived peptideSIINFEKL (SEQ ID NO: 4), as detected in flow cytometry after incubationwith equal amounts

(500 nM) of OVA or Anx-OVA for 12 h. N=3

FIG. 2 shows that incubation of DC with a fusion protein containing theannexin core domain and the model antigen ovalbumin (Anx-OVA) leads tostrongly enhanced CD8+ T cell activation compared to incubation with theantigen OVA alone. A) Schematic presentation of the experiment. Murinebone marrow derived DC were incubated with OVA or Anx-OVA. CD8+ T cellactivation was detected using CD8+ OT-I T cells that carry a transgenicT cell receptor specific for the OVA-derived SIINFEKL (SEQ ID NO: 4)peptide. T cell activation was detected by secretion of Interferon-©(IFN-©). B) Murine bone marrow derived DC were incubated with equalamounts of OVA or Anx-OVA, or with purified SIINFEKL (SEQ ID NO:4)-peptide as positive control. After 12 h of incubation, DC wereco-cultured for further 3-5 days with OT-IT cells. OT-I T cellactivation was detected by measuring IFN-© secretion in ELISA. N=3

FIG. 3 shows that incubation of DC with a fusion protein containing theannexin core domain and the model antigen ovalbumin (Anx-OVA) leads tostrongly enhanced CD4+ T cell activation compared to incubation with theantigen OVA alone. A) Schematic presentation of the experiment. Murinebone marrow derived DC were incubated with OVA or Anx-OVA. CD4+ T cellactivation was detected using CD4+ OT-II T cells that carry a transgenicT cell receptor specific for the OVA-derived ISQAVHAAHAEINEAGR (SEQ IDNO: 5) peptide, T cell activation was detected by secretion ofInterleukin-2 (IL-2). B) Murine bone marrow derived DC were incubatedwith equal amounts of OVA or Anx-OVA. After 12 h of incubation, DC wereco-cultured for 1 day with OT-II T cells. OT-II T cell activation wasdetected by measuring IL-2 secretion in ELISA.

FIG. 4 shows that incubation of DC with a fusion protein containing theannexin core domain and the model antigen ovalbumin (Anx-OVA) leads tostrongly enhanced CD4+ T cell activation compared to incubation with theantigen OVA alone. A) Schematic presentation of the experiment. Murinebone marrow derived DC were incubated with OVA or Anx-OVA. CD4+ T cellactivation was detected using CD4+ OT-II T cells that carry a transgenicT cell receptor specific for the OVA-derived ISQAVHAAHAEINEAGR (SEQ IDNO: 5) peptide, T cell activation was detected by secretion ofInterferon-γ (IFN-γ). B) Murine bone marrow derived DC were incubatedwith equal amounts of OVA or Anx-OVA. After 12 h of incubation, DC wereco-cultured for further 3-5 days with OT-II T cells. OT-II T cellactivation was detected by measuring IFN-γ secretion in ELISA.

FIG. 5 shows that various annexins bind to the receptor LRP-1 with highaffinity. Binding of the indicated recombinant annexins and the annexinA1 core domain to immobilized LRP-1 was detected by quartz crystalmicrobalance. Recombinant annexins were analyzed at 3 differentconcentrations. Depicted are fitted binding curves of the indicatedannexins and the annexin A1 core domain to LRP-l. The calculatedaffinities for all annexins and the core annexin A1 domain range from50-300 nM. Murine annexin A1 (mAnxA1): filled circles; murine annexin A1core domain (mAnxA1 core): open circles; murine annexin A5 (mAnxA5):filled squares; murine annexin A13 (mAnxA13): open squares.

FIG. 6 shows that several annexins bind to the receptor Dectin-1 withhigh affinity. A) Analysis of the binding of recombinant annexin A1(Annexin I) and annexin A5 (Annexin V) to the indicated, immobilizedC-type lectin molecules in ELISA. B) Surface plasmon resonancespectroscopy sensorgrams of the binding of murine annexin A1, annexinA5, annexin A13 and the annexin A1 core domain to the surface moleculeDectin-1. The indicated concentrations of the indicated recombinantannexins were allowed to bind to immobilized Dectin-1 and boundmolecules were measured by surface plasmon resonance. Annexin affinitiesto Dectin-1 were calculated to be in the nanomolar range (˜100 nM).

FIG. 7 shows the domain structures of representative annexin proteins.Orthologs of the 12 human annexins shown in other vertebrates have thesame structures, with strict conservation of the four repeats in thecore region (black) and variation in length and sequence in theamino-terminal regions (shaded). Human ANXA1 and ANXA2 are shown asdimers, with the member of the S100 protein family that they interactwith. Domain structures for other model organisms are derived frompublic data made available by the relevant genome-sequencing projects.Features: S100Ax, sites for attachment of the indicated member of theS100 family of calcium-binding proteins; P, known phosphorylation sites;K, KGD synapomorphy (a conserved, inherited characteristic of proteins);I, codon insertions (+x denotes the number of codons inserted); S-A/b,nonsynonymous coding polymorphisms (SNPs) with the amino acid in themajor variant (A) and that in the minor variant (b); N, putativenucleotide-binding sites; D, codon deletions (−x denotes the number ofcodons deleted); A, alternatively spliced exons; Myr, myristoylation.The total length of each protein is indicated on the right. Taken fromMoss and Morgan. The annexins. Genome Biol. 2004; 5(4): 219.

FIG. 8 shows the accession numbers in FASTA format and an alignment ofthe protein sequences of human and murine annexins A1, A5 and A13. Thesequence identifiers are as follows for the respective sequences.

GI I 47115305 I emb I CAG28612.1 I (hANXA1): SEQ ID NO: 1

GI I 49456639 I emb I CAG46640.1 I (hANXA5): SEQ ID NO: 2

GI I 49456633 I emb I CAG46637.1 I (hANXA13): SEQ ID NO: 3

GI I 71059925 I emb I CAJ16506.1 I (hANXA1): SEQ ID NO: 6

GI I 13277612 I gb I AAH03716.1 I (hANXA5): SEQ ID NO: 7

GI I 13397933 I emb I CAC34623.1 I (hANXA13): SEQ ID NO: 8.

The conserved sequence of the core domain of the annexins is boxed. An *(asterisk) indicates positions which have a single, fully conservedresidue. A : (colon) indicates conservation between groups of stronglysimilar properties. A . (period) indicates conservation between groupsof weakly similar properties.

FIG. 9 demonstrates that vaccination with a fusion protein containingthe annexin core domain and the model antigen ovalbumin (Anx-OVA)strongly improves vaccination efficacy compared to antigen OVA alone. A)Schematic presentation of the experiment. C57BL/6 wt mice were immunizedwith 400 pMol OVA or Anx-OVA per animal. Induction of antigen(OVA)-specific CD8+ T cells was detected 7 days after vaccination usingfluorescently labeled SIINFEKL(SEQ ID NO: 4)—MEW class I tetramers. B)and C) Results indicating the frequency of OVA-specific CD8+ T cellswithin all CD8+ T cells after indicated vaccinations as average of 3mice per group (B) and for each animal individually (C). OVA: ovalbumin,Anx-OVA: Fusionsprotein containing the annexin core domain, a linkingsequence and ovalbumin, −: no vaccination

FIG. 10 shows the DNA-sequence [SEQ ID NO: 13] (A) and amino acidsequence [SEQ ID NO: 14] (B) of the Anx-OVA fusionprotein used forvaccination. light grey shading: human Annexin A1-core domain; noshading: linker sequence; dark grey shading: ovalbumin (OVA).

SEQ ID Nos. 1 to 3 and 6 to 8 show the sequences of the human and mouseannexin 1, 5, and 13, respectively, as used in the context of thepresent invention.

SEQ ID Nos. 4 and 5 show peptide sequences as used in the context of thepresent invention.

SEQ ID Nos: 9 to 12 show primer sequences as used in the presentinvention.

EXAMPLES

Sequences

The sequences are as follows:

UniProt SeqID Protein ID Range referred to in the text 1 P04083 41-344human Annexin A1 core domain 2 P08758 14-317 human Annexin A5 coredomain 3 P27216 13-316 human Annexin A13 core domain 4 P01012 257-267 ova peptide SIINFEKL 5 P01012 323-339  ova peptide ISQAVHAAHAEINEAGR 6P10107 41-344 murine Annexin A1 core domain 7 P48036 12-315 murineAnnexin A5 core domain 8 Q99JG3 14-317 murine Annexin A13 core domain

Mice.

C57BL/6 mice were purchased from the Jackson Laboratory. All mice weremaintained in specific-pathogen-free facilities.

Cells.

For differentiation of BM precursors to BMDCs using recombinant murineGM-CSF, 1×10⁶ cells were seeded at a density of 1×10⁶ cells/ml in RPMI1640 complete medium (10% FCS, 10 U/ml penicillin/streptomycin, 300 mg/lL-glutamine, 20 ng/ml GM-CSF (Immunotools)) in a 24-well plate. After 2days the medium was replaced by fresh medium. After 4 d, half of themedium was removed and replaced by fresh medium. Experiments wereconducted 7-8 d after differentiation.

Generation of Recombinant Core Domain-Antigen Fusionprotein.

The mouse (m)AnxA1-OVA-pET41a plasmid was generated by cloning chickenOvalbumin (OVA; NM_205152 or NP_990483, respectively, from amino acid140) into a modified version of pET41a harboring a C-terminal FLAG tag,a PreScission Protease cleavage site, and a protein A tag. In addition,two flexible linkers and a Tobacco Etch Virus (TEV) cleavage site wereintroduced between mAnxA1 and OVA. Successive PCRs were performed usingthe following primers:

Fw_1: (SEQ ID NO: 9) 5′ GGCGGAGGTTCAGGCGGAGGTTCAGATCAAGCCAGAGAGCTCATC 3′;, Fw_2: (SEQ ID NO: 10) 5′GAAAACTTGTATTTCCAGGGCGGCGGAGGTTCAGGCG 3′;, Fw_3: (SEQ ID NO: 11) 5′GGATCCGGCGGAGGTTCAGGCGGAGGTTCAGAAAACTTGTAT TTCCAGGGCGG 3′ and Rev:(SEQ ID NO: 12) 5′ GGATCCAGGGGAAACACATCTGCCAAAG 3′.

The final PCR product was subcloned using the pGEM®-T easy vector systemfrom Promega. Escherichia coli BL21(DE3)pLysS strain (Promega) was usedto express the fusion-protein. Overnight cultures of E. coli transformedwith the vector described above were used to inoculate 4 L of LBcontaining 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Cultureswere agitated at 180 rpm until A600 nm reached 0.6. Expression wasinduced using 1 mM isopropyl-D-thiogalactopyranoside (IPTG) for 4 hrs at37° C. Cells were harvested by centrifugation and stored frozen at −20°C. Cell pellets containing Protein A-tagged recombinant fusion proteinwere resuspended in native bacterial lysis buffer and disrupted by sixcycles of freeze and thaw. Cell extract was loaded onto IgG Sepharose 6Fast Flow beads (GE Healthcare). Removal of LPS was achieved by washingwith TBS containing 0.1% Triton X-114 (Sigma-Aldrich) as describedpreviously (Reichelt, Schwarz et al. 2006, Zimmerman, Petit Frere et al.2006). Triton X-114 was removed by washing with TBS containing 0.05%Tween-20. After cleavage of the fusion protein with PreScission Protease(GE Healthcare) and removal of PreScission Protease using GlutathioneSepharose Beads 4B (Amersham Biosciences), the recombinant protein wasdialyzed against PBS. After sterile filtration, protein concentrationwas measured using BCA-Assay (Pierce) and LPS-content was determinedusing Limulus Amoebocyte Lysate Assay (Lonza).

Recombinant proteins were expressed in the Escherichia coliBL21(DE3)pLysS strain (Promega) from the pET41a vector (Novagen). PCRproducts encoding a fusionprotein of the annexin A1 core domain and fulllength chicken ovalbumin were cloned into pET41a harboring a C-terminalFLAG_tag, a PreScission protease cleavage site and a Protein A_tag.Bacterial lysates (10,000×g, 4° C. for 40 min) were loaded ontopre-equilibrated IgG Sepharose 6 Fast Flow beads (GE Healthcare).Removal of LPS was achieved by washing with TBS containing 0.1% TritonX-114 (Sigma). Triton X_114 was removed by washing with TBS containing0.05% Tween-20 (Gerbu). After cleavage of the fusion protein withPreScission protease (GE Healthcare) and PreScission protease removal,the recombinant protein was dialysed against PBS. LPS content in allannexin A1 preparations was determined to be below 5 EU/mg using theLimulus Amoebocyte Lysate Assay (Lonza) according to the manufacturers'instructions.

Detection of Antigen Presentation In Vitro.

2×10⁵ BMDCs from C57BL/6 wildtype mice were incubated with 500 nM or theindicated amount of recombinant Ovalbumin (OVA, Sigma) or annexin coredomain-OVA fusionprotein. After 8-12 h, DC were washed with PBS andincubated with a fluorescently labeled antibody against the OVA-derivedpeptide SIINFEKL (SEQ ID NO: 4) in MEW class I (antibody 25-D1.16,eBioscience). SIINFEKL(SEQ ID NO: 4)-positive cells were detected inFACS (FACS-Canto, Becton Dickinson).

Coculture of DC and T Cells and T Cell Activation.

2×10⁵ BMDCs from C57BL/6 wildtype mice were incubated with 500 nM or theindicated amount of recombinant Ovalbumin (OVA, Sigma) or annexin coredomain-OVA fusionprotein. After 12 h, 1×10⁶ magnetically purified(Easysep, Stemcell Technologies) CD8+ or CD4+ T cells from spleens ofOT-I or OT-II mice, respectively, were added to the DC cultures. After1-2 days (Interleukin-2) or 3-5 days (Interferon-γ) indicated cytokineswere determined in the culture supernatants by ELISA (Becton-Dickinson).

Measuring the Affinity of the Binding Between Annexin and LRP-1.

For measuring the affinity of the binding of LRP-1 to different annexins(annexin A1, A5, and A13) the device A100 (ATTANA) was used. LRP-1 wasimmobilized on an LNB carboxychip according to the manufacturers'instructions. In order to achieve this, first, the chip was activatedwith EDC/SulfoNHS according to the manufacturers' instructions, and thenpurified LRP1 (5-15 μg/ml) in a sodium acetate buffer (pH 4.0) wasinjected onto the chip until an increase of the frequency at 70-100 Hzwas reached. Then, remaining binding spots on the chip were saturatedusing two injections of ethanolamine, and the chip was buffered in PBS.For the incubation with the different annexins, they were prepared insix different concentrations in PBS with 2 mM calcium, and measured intriplicates. After each Anx-injection the chip was regenerated with 5 mMEDTA/PBS and 3M NaCl before the next Anx-injection.

Annexin Binding Measurement for Different Receptors by ELISA.

To test for binding to annexins, putative receptor molecules, fragmentsthereof or fusion proteins (e.g. LRP-11, single LRP-1 domains orDectin-1 Fc protein) are immobilized on an ELISA plate at 10 μg/ml incoating buffer (Carbonate-Bicarbonate—1.5 g Na₂ CO₃; 2.93 g NaH CO₃;Distilled water, 1 liter, pH to 9.6). After washing (3×PBS Tween 0.01%)and blocking (1% Casein in PBS), different concentrations of recombinantannexins are incubated in the wells for 2 h, followed by 5 wash steps(PBS-Tween 0.05%). Bound annexins are then detected by suitablesecondary reagents (e.g. horse radish peroxidase (HRP) labeled secondaryantibodies or biotin-labeled secondary antibodies plusstreptavidin-labeled HRP) to the recombinant annexin-proteins andmeasured by reactivity with a suitable substrate (e.g. OPD) in an ELISAplate reader. The assay can also be performed by immobilizing differentannexins on a plate and probing with recombinant receptor molecules,fragments thereof or fusion proteins (e.g. LRP-11, single LRP-1 domainsor Dectin-1 Fc protein).

Binding Affinity Measurements for Annexin-Dectin 1 by Surface PlasmonResonance.

Surface plasmon resonance (SPR) is a valuable tool for analyzingreceptor ligand interactions in real time and for providing insightsinto the affinity and kinetics of binding. SPR is a technique formeasuring the association and dissociation kinetics of ligand, termedanalyte, with a receptor. The analyte or the receptor can be immobilizedon a sensor chip which bears a gold film. The association of the analyteand receptor with one or the other, depending which one is immobilized,induces a change in the refractive index of the layer in contact withthe gold film. This is measured as a change in the refractive index atthe surface layer and is recorded as the SPR signal in resonance units(RU). For the preparation of Dectin-l-coated surfaces, Dectin-1 wasimmobilized at a flow rate of 10 μl/min. The CMS chip was activated byinjection of a mixture of N-ethyl-N′-(diethylaminopropyl)-carbodiimide(EDC) and N-hydroxysuccinimide (NHS) for 10 minutes and functionalizedby injecting 100 μg/mL and 10 μg/mL Dectin-1 in acetate buffer pH 5.5for 7 minutes. The remaining activated carboxyl groups were then cappedby injection of 1 M ethanolamine for 10 minutes. Control flow cells weretreated with EDC/NHS followed by ethanolamine as described.Concentration gradients of the different annexins were injected over theDectin-l-functionalized surfaces at 10 μL/min, allowing 60 seconds forcontact and 300 seconds for dissociation times, followed by regenerationusing 100 mM methyl-α-D-mannopyranoside at 30 μL/min for 30 seconds.Experimental data were analyzed using Biacore S20 T100 EvaluationSoftware. Kinetic analyses based on a 1:1 interaction model for theannexin-dectin-1 complexes interaction were performed using Scrubber2(BioLogic Software, Campbell, Australia).

In FIG. 9 an in vivo experiment demonstrates that vaccination with afusion protein containing the annexin core domain and the model antigenovalbumin (Anx-OVA) strongly improves vaccination efficacy compared toantigen OVA alone.

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The invention claimed is:
 1. A method for treating or preventing aninfectious disease or cancer in a subject, comprising: administering, tosaid subject, a pharmaceutical composition comprising an effectiveamount of a fusion protein, and a carrier, wherein the fusion proteincomprises (i) at least one annexin core domain, wherein the annexin coredomain consists of a sequence selected from: (a) amino acids 41-344 ofSEQ ID NO: 1, (b) amino acids 14-317 of SEQ ID NO: 2, (c) amino acids13-316 of SEQ ID NO: 3, (d) amino acids 41-344 of SEQ ID NO: 6, (e)amino acids 12-315 of SEQ ID NO: 7, and (f) amino acids 14-317 of SEQ IDNO: 8, or wherein the annexin core domain consists of an amino acidsequence that is at least 80% identical to the sequence selected from(a) to (f), and (ii) at least one antigenic peptide that is presented bya major histocompatibility complex (MHC).
 2. The method of claim 1,wherein the at least one antigenic peptide is derived from an antigenselected from the group consisting of βhCG, gp100, Pmel17, HER2/neu,WT1, mesothelin, CEA, MART1, TRP-2, NY-BR-1, NY-CO-58, MN (gp250),idiotype, tyrosinase, telomerase, SSX2, MUC-1, MART1, melan-A, NY-ESO-1,MAGE-1, MAGE-3, MAGE-A3, and high molecular weight-melanoma associatedantigen (HMW-MAA).